Using T cell recognition (particularly cell-mediated cytotoxicity) we have continued to probe the complexities of the alloantigeni differences between normal human donors. In contrast to our previous studies trying to induce T cell responses against new determinants, we are analyzing, at the molecular level, the specificity of 10 'DPw2-specific' cytotoxic T lymphocyte (CTL) clones already generated. Analysis of their specificity on panels of MHC-loss mutant lymphoblastoid cell lines has been extended. These studies confirm the identification of CTL-defined differences between mutant cell lines which is not explained by differences in the DP gene, but rather appear to reflect mutation of another gene within HLA. Biochemical analysis suggests that the differeneces correlated with subtle differences on 2-dimensional gel electrophoresis in the Ia-related molecules precipitated by an anti-invariant chain antibody. We hypothesize that hybrid molecules (e.g., DP-beta + DR-alpha) may contribute to this recognition. Northern blot analysis is in progress to test the hypotheses that differences in expression of DRor DQ genes will correlate with these differences in recognition. Two other genetic approaches are being used to analyze the target cell requirements for killing by these CTL clones. First, mutant cell lines are being isolated by mutagenesis and negative selection with the CTL clones themselves (rather than with antisera). Of two mutants which have been derived and are being analyzed in detail, both have lost the capacity to be recognized by DPw2-specific CTL, but only one has lost serologic expression of DPw2. The genetic basis for these alterations are under investigation. Second, cells are being transfected with class II genes including those thought to encode DPw2. Our findings indicate that reconstitution of function as a target cell for DPw2-specific CTL requires more than expression of the DPw2 molecule. These additional requirements are being defined.